Synthesis of steroids by cylindrocarpon radicicola



United States Patent This application is a division of a prior application, Serial No. 165,657, filed January 11, 1962, now U.S. Patout No. 3,174,982.

This invention relates to the synthesis of new steroids and derivatives thereof, and more particularly, to the preparation of new testololactones and secoandrostatrienoic acids.

The final compounds of this invention are of the general formula wherein X is hydrogen, chloro or fluoro (preferably hydrogen or fluoro), and R is hydrogen or a hydrocarbon radical of less then ten carbon atoms, as exemplified by lower alkyl (e.g., methyl, ethyl, propyl, butyl, hexyl and octyl), monocyclic ar(lower alkyl) (e.g., benzyl, phenethyl, 3-phenylpropyl, 4-phenylbutyl, and o, m, or p-tolylethyl), monocyclic aryl, cycloalkyl, cycloalkenyl, cycloalkyl(lower alkyl), lower alkenyl, and monocyclic ar(lower alkenyl).

The final compounds of this invention, wherein R is hydrogen, can be obtained directly by culturing Cyiindrocarpon radicicola in the presence of either ll ketoprogesterone or a 9ot-halo-ll-ketoprogesterone, whereby a mixture of the desired final product and either ll-ketol-dehydrotestololactone or 9a-fluoro (or chloro)-11-ketol-dehydrotestololactone is produced; or indirectly from said 1-dehydrotestololactones by treatment of the latter with a base.

The method of culturing Cylindrocarpon radicicola for the purpose of this invention is the same as that described in US. Patent No. 2,868,694, except that either ll-ketoprogesterone or a 9a-halo-1l-ketoprogesterone is employed as the steroid substrate. That is, the steroid is added to a growing culture of Cylindrocarpon radicicola either during the incubation period or by including it in the nutrient medium prior to inoculation. If ll-ketoprogesterone, 9Dt-lOdO-1 l-ketoprogesterone, or 9oc-bfOII10- ll-ketoprogesterone is employed as the substrate, A 13,17-seco-androstatriene-3,1l-dione-17-oic acid is obtained as the product. If, however, 9a-fluoro-ll-ketoprogesterone is used as the substrate, 90t-fillOlO-A 13,l7-seco-androstatriene-3,l1-dione-17-oic acid is obtained; and 9oc-chloro-1l-ketoprogesterone yields 9a-chloro-A -13,l7-seco-androstatriene-3,11-dione-l7oic acid.

These free acids can then be converted to their ester derivatives in the usual manner, as by treatment with a desired alcohol in the presence of an esterification catalyst or, if a lower alkyl ester is desired, by treatment with a diazo(lower alkane) such as diazomethane.

The final compounds of this invention are physiologically active substances which possess protein-anabolic activity, and hence can be used in lieu of known proteinanabolic steroids, and may be administered either perorally or parenterally in the treatment of post-operative shock and other conditions where tissue degeneration has occurred, being formulated for such administration in the same type of preparation as testosterone, for example with concentration and/or dosage based on the activity of the particular compound.

As stated hereinbefore, in addition to the secoandro statrienoic acids, the process of this invention employing Cylindrocarpon radz'cicola as the microorganism yields 11 keto 1 dehydrotestololactone, chloro 11- keto-l-dehydrotestololactone, or 9a-fluoro-11-keto-l-dehydrotestololactone, depending on whether the steroid substrate is ll-ketoprogesterone (or 9a-iodo-11-ketoprogesterone or 9a-bromo-ll-ketoprogesterone), 9tx-chloro-11- ketoprogesterone, or 9a-fiuoro-ll-ketoprogesterone, respectively.

These 11-keto-1-dehydrotestololactones are new compounds of this invention which may be converted to the final products of this invention by treatment with a strong base, such as an alkali metal hydroxide (e.g., potassium hydroxide and sodium hydroxide). The reaction is preferably conducted at an elevated temperature.

The 11-keto-l-dehydrotestololactone intermediates of this invention can also be prepared by a two-step process of this invention. In the first step, ll-ketoprogesterone, 9ix-chloro-l1-ketoprogresterone or 9a-fluoro-11-ketoprogesterone is subjected to the action of Penicillium citrinum, whereby ll-ketotestololactone, 9e-chloro-ll-ketotestololactone, and 9ix-fluoro-1l-ketotestololactone are formed, respectively. These ll-ketotestololactones are new intermediates of this invention.

The conversion is effected by either bringing together, in an aqueous medium, the steroid, oxygen, and enzymes of non-proliferating cells of Penicillium citrinum or, preferably, by including the steroid in an aerated culture of Penicillium citrinum.

When aerated culture is used, the oxidation is eifected in the presence of Penicillz'um citrznum by adding the steroid to the culture during the incubation period, or by including it in the nutrient medium prior to inoculation. In any case, assimilable sources of nitrogenous materials for growth promotion and carbon-containing substances as energy sources should be present in the culture medium. Also, an adequate air supply should be maintained during the oxidation, e.g., by the conventional techniques of (1) exposing a large surface of the medium to air or (2) aerating in submerged culture.

In general, the condition of culturing Penicillium citrinum for the purpose of this invention are (except for the inclusion of the steroid) the same as those of culturing such microorganism for the production of other metabolites. Thus, the nutrient medium essentially comprises assimilable sources of nitrogen for growth and carbon for energy.

The nitrogen source materials may be organic (e.g., soybean metal, cornsteep liquor, meat extract, and/ or distillers solubles) or synthetic (i.e., composed of simple, synthesizable organic or inorganic compounds such as ammonium salts, alkali nitrates, amino acids, urea 01' thiourea).

As to the energy source material, lipids, especially (1) fat acids having at least 14 carbon atoms, (2) fats or (3) mixtures thereof, are preferred. Examples of such fats are lard oil, soy-bean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein and trilaurin; and illustrative fat acids include stearic, palmitic, oleic, linoleic and myristic acids.

Other carbon-containing materials may also be used. For example such materials as glycerol, glycose, fructose,

sucrose, lactose, maltose, dextrins, starches, whey, etc., are adequate carbon source materials. These materials may be used either in purified state or as concentrates, such as whey concentrate, corn, wheat or barley mash; or mixtures of the above may be employed. It is to be noted, however, that the steroid is added to the fermentation medium essentially as a. precursor and not as an energy source.

The ll-ketotestololactones thus formed can then be dehydrogenated in the 1,2-position, either microbiologically, as by treatment with a known l-dehydrogenating microorganism such as N ocardia restrictus, or chemically, as by treatment with selenium dioxide, or 2,3-dichloro- 5,6-dicyanobenzoquinone to yield the ll-keto-l-dehydrotestololactones of this invention.

In addition to their use as intermediates, the ll-ketotestololactones of this invention, that is compounds of the general formula wherein the 1,2-position is saturated or double-bonded, and X is as hereinbefore defined, are physiologically active substances which possess protein-anabolic activity, and hence can be used in lieu of known protein-anabolic agents in the treatment of osteoporosis, protein tissue, depletion and chronic de'bility and tissue atrophy in geriatric patients, for which purpose they are administered either perorally or parenterally in the treatment of post-operative shock and other conditions where tissue degeneration has occurred, being formulated for such administration in the same type of preparations as testosterone, for example, with concentration and/or dosage based on the activity of the particular compound.

The following examples illustrate the invention (all temperatures being in centigrade) Example l.-9a fluoro-II-keto-J-dehydrtest0l0laclone and 9a fluoro A -13,17-sec0-andr0statriene-3,11- di0ne-l7-0ic acid Surface growth from a three-week old agar slant culture of Cylindrocarpon radicicola (ATCC 11011), the slant containing as a nutrient medium (A): glucose, g.; Difco yeast extract, 2.5 g. K I-IPO l g.; agar, g.; and distilled water to 1 l., is suspended in 2.5 ml. of a 0.01% sodium lauryl sulfate aqueous solution. One milliliter portions of the suspension are used to inoculate two 250 ml. conical flasks, each containing 50 ml. of the following sterilized nutrient medium (B): dextrose, 10 g.; cornsteep liquor, 6 g.; NH H PO 3 g.; Difco yeast extract, 2.5 g.; CaCl 2.5; and distilled water to 1 1. After forty-eight hours of incubation at with continuous rotary agitation (280 cycles per minute, 2 inch radius), 10% (vol./vol.) transfers are made to twelve 250 ml. conical flasks each containing 50 ml. of fresh sterilized medium B. These are incubated under the conditions described above for twenty-four hours, after which another 10% (vol/vol.) transfer is made to 100 additional 250 ml. flasks containing 50 ml. of fresh sterilized medium E. The 9a-fluoro-1l-ketoprogesterone is added by adding to each flask 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide (60 mg./ml.) so that the medium is supplemented with 300 ag/ml. of steroid. After 48 hours of further incubation, the contents of the flasks are pooled and filtered through a Seitz clarifying pad. The flasks, mycelium and pad are washed with successive 50 ml. portions of warm water. The combined filtrate and washings (pl-I 8.2) are acidified to pH 4 with acetic acid and extracted three times with 2 1. portions of chloroform. The combined chloroform extracts are then washed twice with 3 1. portions of water and evaporated to dryness, in vacuo. The residue is redissolved in 200 ml. of ethyl acetate and extracted with 2 x ml. portions of 5% sodium bicarbonate. The ethyl acetate is then washed with water, dried over sodium sulfate and evaporated to dryness, in vacuo. Crystallizations of the residue from acetone-hexane gives about 100 mg. of 9a-fluoro-11- keto-d -dehydrotestololactone having a melting point of about 217220; +285 (chlf.);

x i 234. um (e=16,000); mi? We WI 241 ma (6: 27,400); A235,? 5.80, 5.86, 6.00, 6.13, 622 E33 1.19 volts (vs. mercury pool anode).

Analysis.-Calcd. for C H O F (332.48): C, 68.66; H, 6.37. Found: C, 68.95; H, 6.12.

The sodium bicarbonate extracts are acidified with 2 N hydrochloric acid and extracted with three 100 ml. portions of chloroform. The combined chloroform extracts are then washed with water and evaporated to dryness, in vacuo. Crystallization of the residue from acetonehexane gives about 910 mg. of 9a-fluoro-A -13,17- seco-androstatriene-3,11-dione-17-oic acid having a melting point of about 217220; [@1 3 46.5 (chl=f.);

Mir... 240 m (c=28,500) x3333? 5.80, 6.02, 0.12, 6.24 EBQ 1.14 volts (vs. mercury pool anode).

Analysis.--Calcd. for C, H O F (332.48): C, 68.66; H, 6.37. Found: C, 69.03; H, 6.21; Neut. Eq. 347.

Example 2 The procedure of Example 1 is followed except that the final stage is carried out in 2 l. flasks containing 500 ml. of medium and the pH is adjusted daily with hydrochloric acid to a value slightly less than 7. The combined filtrate and washings are then extracted as described in Example 1 and separated into the acidic and neutral fractions. Crystallization of each gives respectively about 360 mg. of 9a-fiuoro-A -13,17-seco-androstatriene-3,11-dione-17-oic acid and about 510 mg. of 9ot-fluoro-11-keto-A -dehydrotestololactone.

Example 3 9a-chl0r0-11-ket0-1-dehydr0testol0lact0ne and 9a-chl0r0-A -1 3 ,1 7-sec0-andr0statrierte-3,] l-di- 0ne-17-oic acid Following the procedure of Example 1 but substituting an equivalent amount of 9tx-chloro-1l-ketoprogesterone for the 9a-fluoro-1l-ketoprogesteroue, 9a-chloro-11-ketol-dehydrotestololactone and 9a-chloro-A -13,17-secoandrostatriene-3,11-dione-l7-oic acid are obtained.

Example 4.-11-keto-1-dehydrotestololactone and A 13,17-sec0-andr0statriene-3,1I-dione-l 7 -0ic acid Following the fermentation and extraction procedure described in Example 1 but substituting ll-ketoprogesterone for the 9a-fluoro-ll-ketoprogesterone, there is obtained about 320 mg. of 11-keto-A -dehydrotestololactone having M.P. about 196-198"; [(11 +79.0 (chlf.);

A12 238 m (e=15,900); k211i?" 239 m (6 25,600); A232" 5.74, 5.83, 6.00, 6.13, 622a.

Analysis.Calcd. for C H O (314.37): C, 72.59; H, 7.05. Found: C, 72.51; H, 7.24 and about 363 mg. of A -13,l7-seco-androstatriene 3,11 dione-17-oic acid having M.P. about 167-169; [M i0 (chlf.);

Example 5 The fermentation is carried out as described in Example I except that 9a-bromo-1l-ketoprogesterone is used as the substrate and the final stage of the fermentation is carried out in 2 l. flasks containing 500 ml. of medium. The products are separated into neutral and acidic fractions as described in Example 1 to give upon crystallization about 500 mg. of 1l-keto-A -dehydrotestololactone and about 200 mg. of A -13,17-seco-androstatriene- 3,11-dione-17-oic acid identical with the products of Example 4.

Similarly, 9a-iodo-1l-ketoprogesterone yields ll-keto- A -dehydrotestololactone and A -13,17-seco-androstatriene-3,1l-dione-17-oic acid.

60 mg. of 9a-fluoro-11-keto-A -dehydrotestololactone is dissolved in 5 ml. of 5% potassium hydroxide in methanol and the solution is warmed on a steam bath for five minutes. It is then cooled, neutralized with acetic acid, diluted with water and extracted with ether. The ether is then re-extracted with 5% sodium bicarbonate which is acidified and extracted with ether. The ether is dried and evaporated to dryness and the residue crystallized from acetone-hexane to give 9a-fluoro-A -13,17-seco-androstatriene-3,11-dione-17-oic acid.

Analysis.-Calcd. for C H O F (332.48): C, 68.64; H, 6.37; F, 5.71. Found: C, 67.78; H, 6.81; F, 5.67.

Example 7.A -13,Z 7-sec0-andr0statriene-3,1J-dione- 17-01'0 acid Following the procedure of Example 6, but substituting an equivalent amount of 1l-keto-n -dehydrotestololactone for the 9a-fluoro-11-keto-A -dehydrotestololactone, N 13,17-seco-androstatriene-3,11-dione-17-oic acid is obtained.

To a suspension of 360 mg. of 9a-fluoro-A -13J7- seco-androstatriene-3,11-dione-17-oic acid in 10 ml. of methanol, an ethereal solution of diazomethane is added slowly with swirling until the steroid dissolved and the yellow color of the diazomethane persists. After 30 minutes at room temperature the solution is evaporated, in vacuo, and the residue distributed between ethyl acetate and 5% sodium bicarbonate. The ethyl acetate is Washed well with Water, dried over sodium sulfate and evaporated to dryness, in vacuo. Chromatography of the residue on Woelm neutral alumina and elution with chloroformbenzene (1:9) gives on crystallization from acetone-hexane about 200 mg. of methyl 9a-fluoro-A -13,17-secoandrostatriene-3,l1-dione-l7-oate, melting point about 119-121; 46.9 (chlf.);

k 239 m (e=29,400); k113i?5.80,.5.966.01,6.12,6.21;l

Analysis.-Calcd. for C H O F (346.38): C, 69.34; H. 6.69; F, 5.49. Found: C, 69.50; H, 7.09; F, 5.53.

Example 9.M ethyl A -13,17-sec0-andr0statriene- 3,11-di0ne-1 7-0ate Example 1 1 .1 1 -ketotest0l0lact0ne Following the fermentation and extraction procedures described in Example 1 but substituting Penicillin citrium 6 (ATCC 8506) for C. radicicola and ll-ketoprogesterone for 9a-fluoro-1l-ketoprogesterone there is obtained about 650 mg. of ll-ketotestololactone having a melting point of about 226228, [a] +121 (chlfi);

max.

Analysis.-Calcd. for C H O (316.38); C, 72.12; H, 7.65. Found: C, 72.26; H, 7.77.

Example 12.9a-fluoro-l1-ket0-A -dehydrotestololactone The fermentation is carried out as described in Example 1, however, a culture of Nocardia restrictus (Culture Collection, Rutgers Institute of Microbiology, No. 545) is substituted for the C. radicicola and 200 mg. of 9a-fiuoroll-ketotestololactone is used as the substrate and distributed in twenty 250 ml. flasks. The fermentation is allowed to proceed for 7%. hours, then filtered and washed With water and the combined filtrate and washing (1.1 l.) extracted three times with 300 ml. portions of chloroform. The chloroform extracts are combined, washed with Water and evaporated to dryness, in vacuo. Crystallization of the residue from acetone-hexane gives about mg. of 9u-flI1OIO-1 l-keto-A -dehydrotestololactone.

Similarly, 9tx-chloro-1l-ketotestololactone yields 9achloro-l l-keto-A -dehydrotestololactone.

Example 13 .1 1 -ket0-A -dehya'rotestololactone wherein X is selected from the group consisting of hydrogen, chloro and fluoro, which comprises subjecting a compound selected from the group consisting of ll-ketoprogesterone and 9a-halo-1l-ketoprogesterones to the action of enzymes of the microorganism Cylindrocarpon radicicola and recovering the acid formed.

2. A process for preparing a compound of the formula wherein X is selected from the group consisting of hydrogen, chloro and fluoro, which comprises subjecting a compound selected from the group consisting of ll-ketoprogesterone and 9ot-halo-1l-ketoprogesteroues to the action of enzymes of the microorganism Cylindrocarpon radicicola and recovering the testololactone formed.

References Cited by the Examiner UNITED STATES PATENTS 10/1960 Thoma et a1 -51 3/1963 Principe et al 19551 A. LOUIS MONACELL, Primary Examiner.

D. M. STEPHENS, Assistant Examiner. 

1. A PROCESS FOR PREPARING A COMPOUND OF THE FORMULA 1-(HOOC-CH2-CH2-),2-(CH3-),4A-X-1,4,4A,4B,7,9,10,10A-OCTAWHEREIN X IS SELECTED FROM THE GROUP CONSISTING OF HYDROGEN, CHLORO AND FLUORO, WHICH COMPRISES SUBJECTING A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 11-KETOPROGESTERONE AND 9A-HALO-11-KETEPROGESTERONES TO THE ACTION OF ENZYMES OF THE MICROORGANISM CYLINDROCARPON RADICICOLA AND RECOVERING THE ACID FORMED.
 2. A PROCESS FOR PREPARING A COMPOUND OF THE FORMULA 1,2-(-CH2-CH2-COO-),4A-X-1,2,3,4,4A,4B,7,9,10-DECAHYDROPHENANTHRENE-4,7 -DIONE HYDROPHENANTHRENE-4,7-DIONE WHEREIN X IS SELECTED FROM THE GROUP CONSISTING OF HYDROGEN, CHLORO AND FLUORO, WHICH COMPRISES SUBJECTING A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 11-KETOPROGESTERONE AND 9A-HALO-11-KETOPROGESTERONES TO THE ACTION OF ENZYMES OF THE MICROORGANISM CYLINDROCARPON RADICICOLA AND RECOVERING THE TESTOLOLACTONE FORMED. 